Angiogenesis, the sprouting and development of new blood vessels from pre-existing ones, is a fundamental process that occurs in embryonic development and tissue remodelling (Folkman, J., 1995, Nat. Med. 1, 27-31). The development of the neovasculature requires the proliferation and migration of endothelial cells, and their interaction with other cell types including fibroblasts and pericytes to form complex three-dimensional cord-like structure that contain endothelial monolayers surrounding the lumen of the vessel (Risau, W., 1997, Nature 386, 671-674). The mechanisms controlling angiogenesis are not completely understood, but it is likely that the progression and development of new blood vessels is regulated at multiple control sites, and involves growth factors (Meyer, M et al, 1999, EMBO J. 18, 363-374), proteases (Kubota, Y., et al., 1988, J. Cell Biol. 107, 1589-1598), the extracellular matrix (Preissner, K. T., et al., 1997, Thromb. Haemost. 78, 88-95), and the expression of cell-surface receptors (Bazzoni, G., et al., 1999, Curr. Opin. Cell Biol. 11, 573-581). The identification of factors that regulate and control angiogenesis is of increasing clinical importance since inappropriate or insufficient angiogenesis is central to a number of pathological conditions, including the development of solid tumours, rheumatoid arthritis and psoriasis (Folkman, J., 1995, Nat. Med. 1, 27-31).
The search for molecules that modulate angiogenesis is of emerging clinical importance, particularly with respect to solid tumor therapy. Specific inhibition of angiogenesis has the potential to revolutionise cancer therapy, by starving tumours of their blood supply (O'Reilly, M. S., et al., 1997, Cell 88, 227-285), and this approach to cancer therapy led to the discovery of the anti-angiogenic peptides endostatin (O'Reilly, M. S., et at., 1997, Cell 88, 227-285) and angiostatin (O'Reilly, M. S., et al., 1996, Nat. Med. 2, 689). Modulation of angiogenesis also presents the possibility of new treatments for other pathological conditions that rely on vascular remodelling.
Transglutaminases are an important class of protein crosslinking enzymes that catalyze protein aggregation reactions in blood coagulation (Greenberg, C. S., et al., 1991, FASEB J. 5, 3071-3077), skin maturation Thacher, S. M. & Rice, R. H., 1985, Cell 40, 685-695) and the clotting of seminal secretions (Dubbink, H. J., et al., 1999, Lab. Invest. 79, 141-150). The most widespread member of the family is the cellular form of the enzyme, tissue transglutaminase (tTGase), which is expressed in varying amounts in many cell types. Like the well-characterized plasma TGase (blood coagulation factor XIIIa) (Greenberg, C. S., et al., 1991, FASEB J. 5, 3071-3077) and keratinocyte TGase (Thacher, S. M. & Rice, R. H., 1985, Cell 40, 685-695), tTGases are calcium-dependent enzymes that catalyze the formation of crosslinks proteins via ε(γ-glutamyl) isopeptide bonds and the incorporation of polyamines at certain glutamine residues (Greenberg, C. S., et al., 1991, FASEB J. 5, 3071-3077).
However, tTGase is unique in the transglutaminase family of enzymes in that is able to bind and hydrolyze GTP and ATP (Achyuthan, K. B. & Greenberg, C. S., 1987, J. Biol. Chem. 262, 1901-1906), and to bind to fibronectin (Achyuthan, K. E., et al., 1995, J. Immunol. Methods 180, 67-79). The enzyme is predominantly located in the cytosol, although tTGase has also been reported to exist in the nucleus (Lesort, M., et al., 1998, J. Biol. Chem. 273, 11991-11994), at the cell surface and in the extracellular matrix (Martinez, J., et al., 1994, Biochemistry 33, 2538-2545). Tissue TGase is highly expressed in endothelial cells (Greenberg, C. S., et al., 1987, Blood 20, 702-709) and its activity at the surface of such cells is thought to enhance basement membrane stabilisation, cell spreading and cell adhesion (Martinez, J., et al., 1994, Biochemistry 33, 2538-2545, Greenberg, C. S., et al., 1987, Blood 20, 702-709, Kinsella, M. G. & Wight, T. N., 1990, J. Biol. Chem. 265, 17891-17896, Jones, R. A., et al., 1997, J. Cell Sci. 110, 2461-2472, Gaudry C. A., et al., 1999, Exp. Cell Res. 252, 104-113). However, the overall significance of the high amount of enzyme in this cell type and its biological function is poorly understood.
Antisense studies in the endothelial-like cell line ECV304 have demonstrated that tTGase-deficient cells exhibit severely impaired cell adhesion (Jones, R. A., et al., 1997, J. Cell Sci. 110, 2461-2472), and ECV304 cells transfected with a chimeric form of the enzyme that contains a Protein kinase Cε-epitope tag show strong tTGase staining at cell adhesion sites that co-distribute with the β1-integrin (Gaudry C. A., et al., 1999, Exp. Cell Res. 252, 104-113).
Recently, a study by Haroon et al (Haroon, Z. A., et al., 1999, FASEB J. 13, 1787-1795) has demonstrated that tTGase is expressed and active in rat dermal wound healing and angiogenesis. By examining biopsy punch wounds throughout the healing process, tTGase antigen and activity were observed at sites of neovascularisation and in the provision of fibrin matrix in the wound bed 24 hours after injury. Tissue TGase antigen levels increased four- to five-fold at three days after wounding and were ultimately degraded. In this study, it was also found that endothelial cells, macrophages and skeletal muscle cells express tTGase and that the addition of exogenous recombinant tTGase led to an increase in vessel length density.
On the basis of studies such as those by Haroon et al, which highlight a role of TGase in promoting angiogenesis, skilled persons have sought TGase inhibitors as therapeutic agents for inhibiting angiogenesis.